Orosomucoid-1 Expression in Ameloblastoma Variants.

Odontogenic tumors constitute a group of heterogeneous lesions of benign and malignant neoplasms with variable aggressiveness. Ameloblastomas are a group of benign but locally invasive neoplasms that occur in the jaws and are derived from epithelial elements of the tooth-forming apparatus. We previously described orosomucoid-1 protein expression in odontogenic myxomas. However, whether orosomucoid-1 is expressed in other odontogenic tumors remains unknown. Since orosomucoid-1 belongs to a group of acute-phase proteins and has many functions in health and disease, we identified and analyzed orosomucoid-1 expression in ameloblastoma variants and ameloblastic carcinoma using western blot and immunohistochemical techniques. Thirty cases of ameloblastoma were analyzed for orsomucoid-1; five specimens were fresh for western blot study (four benign ameloblastomas and one ameloblastic carcinoma), and 25 cases of benign ameloblastoma for immunohistochemical assays. Orosomucoid-1 was widely expressed in each tumor variant analyzed in this study, and differential orosomucoid-1 expression was observed between benign and malignant tumor. Orosomucoid-1 may play an important role in the behavior of ameloblastomas and influence the biology and development of the variants of this tumor.

dontogenic tumors (OTs) constitute a heterogeneous group of relatively rare benign and malignant neoplasms that display variable aggressiveness (1,2). Ameloblastoma is a benign but locally aggressive OT of the mandible and maxilla that could cause severe facial disfigurement and functional impairment if neglected (3).
Ameloblastoma is thought to arise from epithelial cells in developing teeth, including cells of the dental lamina and enamel organ (4). However, the molecular mechanisms that regulate ameloblastoma cell growth and invasion are unknown. In Mexico, the prevalence of ameloblastoma has been estimated to be 23.7% of all OTs (5). According to Orosomucoid-1 (ORM1) is a 41-43-kDa glycoprotein with a pI of 2.8 to 3.4 that is produced in the liver and secreted into the serum during acute inflammation. ORM1 was described in 1956 by Schmid as a member of a group of acute-phase proteins that might play a role in modulating immune responses to stress, among other functions (9).
Several studies in the literature have described the use of ORM1 in the diagnosis of different cancer types such as bladder, colorectal, or ovarian cancer (10,11).
ORM1 might play a role in defense or resistance mechanisms against tumor cells specifically by reducing the proliferation, invasion, and metastasis of cancer cells, thereby influencing tumor invasion and growth (11). ORM1 inhibits polymorphonuclear neutrophil activation and is therefore considered as a natural immunomodulatory, anti-inflammatory, anti-neutrophil, and anti-complement agent (12).
ORM1 may also suppress lymphocyte and platelet responsiveness by interfering in a common activation pathway. ORM1 may perturb the lymphoid cell surface and interfere with events required for lymphocyte proliferation by altering membrane fluidity and inhibiting concanavalin A receptor and surface immunoglobulin capping (13).
Additionally, in a study on human myocardial infarction, human polymorphonuclear cells were shown to synthesize and release ORM1, suggesting that ORM1 provides endogenous inhibitory feedback in response to excessive inflammation (14).
We previously reported that ORM1 is overexpressed in odontogenic myxomas (15). to better understand the differences in the biological behavior of these tumors.

Western blot
Western blot analysis was performed as previously described (17). Briefly, the proteins in rehydration solution were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes.

Histochemical and immunohistochemical staining
Ameloblastoma specimens were fixed in 10% Immunohistochemical studies were performed as previously described (18) All cases with immunostaining between 10 to 50% were regarded as positive, and all cases with staining>50% were considered highly positive (19).    This table shows the quantification of the positivity for ORM1 by immunohistochemistry. All cases with immunostaining between 10 to 50% were regarded as positive, and all cases with staining > 50% were considered highly positive. More SMA cases had high positivity (> 50%) than UA case, whereas the only AC case was highly positive. UA: unicystic ameloblasoma; SMA: solid multicystic ameloblasoma; AC: ameloblastic carcinoma.

ORM1 expression in ameloblastoma variants and ameloblastic carcinoma
To identify ORM1 protein in tumor samples, we performed Western blot assays using a commercial monoclonal antibody. The antibody strongly recognized a single protein band of approximately 44 kDa, which was the expected molecular weight of ORM1 in all the analyzed samples (Fig. 1). An intense 42-kDa band was also detected in all samples by an anti-actin antibody, which was used as an internal loading control (Fig. 1). We then performed densitometry of the bands detected by the antibodies to semi-quantitatively analyze the relative ORM1 expression in the samples.
Interestingly, ORM1 levels were up to four times higher in AC than in UA or SMA (Fig. 1).
Furthermore, ORM1 was expressed in all of the analyzed samples, but at varying levels.

Confirmation and in situ determination of ORM1 expression in ameloblastoma variants
To determine the in situ ORM1 expression pattern in clinically obtained tumor samples, we performed immunohistochemical assays using a monoclonal ORM1 antibody. ORM1 was expressed in all analyzed samples, particularly in the cytoplasm of epithelial tumor cells, in microcysts from some variants, within the endothelial cells of large and small blood vessels of all samples and within connective tissue stroma of AC and SMA only (Fig. 2, 3, 4).  Differences in ORM1 expression revealed that AC expressed ORM1 in more sites within neoplasms and that ORM1 is more abundant in AC than in SMAs and UAs (Table 1).
Interestingly, ORM1 was expressed in pleomorphic cells in some AC microcysts (Fig. 4A,   B). Additionally, UAs expressed ORM1 in epithelial cells and blood vessels but not in stromal.

cells; in contrast, the stroma of ACs and
SMAs expressed ORM1. Among these cases, SMAs contained more ORM1-expressing cells (Fig.   3A, 4A). Finally, the accumulation of ORM1 in some spaces within SMA microcysts suggests that ORM1 secretion might occur from these structures (Fig. 3A, B). Notably, due to the small number of cases (justified by the rarity of the tumors), only the percentages were described and considered as a trend (Table 1).

Discussion
The acute-phase response is the reaction of an organism to a disturbance in homeostasis and is characterized by dramatic changes in the concentrations of certain plasma proteins, defined as acute-phase proteins (20). ORM1 is an acutephase protein, and increased ORM1 levels have been reported in the serum of patients with various malignant diseases, including hepatocellular carcinoma, gynecological carcinomas, esophageal cancer and head and neck cancers (21)(22)(23)(24). Human hepatocytes, endothelial cells and other cells normally produce ORM1 (21,25,26).
In the present study, ORM1 was expressed in all samples analyzed, suggesting an important role for ORM1 in the development and biological behavior of these tumors. In general, the distribution of ORM1 was the same in all analyzed samples (Fig. 2, 3, 4). However, in UAs, ORM1 was not as clearly expressed in the mesenchymal tissue ( Fig. 2A, B). Moreover, all blood vessels were positive for ORM1.
Suggesting the specific function of ORM1 in ameloblastoma is difficult due to the multiple roles for ORM1 that have been described (9). The high expression of ORM1 can act as a defense mechanism against tumor cell proliferation and invasion. This mechanism has been suggested in colon cancer cells, in which ORM1 overexpression decreased the colony-forming capacity, invasion and adhesion, whereas ORM1 inhibition using antisense oligodeoxynucleotides increased these events (11). Alternatively, given its antiinflammatory activity, ORM1 overexpression could inhibit the immune response, resulting in increased tumor cell proliferation (9). ORM1 may also play an important role in angiogenesis (25), proliferation, invasion and adhesion (11), in the regulation and activation of immune cells, and finally, as a carrier of defense substances (9).
Thus far, background ORM1 expression has not been found in any variant of ameloblastomas and has only been found in odontogenic myxomas (15), but the nature of these tumors is different; ameloblastoma is an epithelial tumor, whereas odontogenic myxoma is a mesenchymal tumor.
It is generally considered that ORM1 is able to inhibit polymorphonuclear neutrophil activation, since it acts as a natural anti-inflammatory, antineutrophil, anti-complement and immunomodulatory agent (12). This leads us to consider a possible immunomodulatory function of ORM1 in the biological behavior of ameloblastomas.
On the other hand, there have been reports stating that ORM-1 by itself increases migration, In a recent work, our group has determined by immunohistochemical technique the presence of VEGF and ORM1 in odontogenic myxomas (29).
All the previously gathered data suggest a possible collaborative pro-angiogenic role of ORM1, but to corroborate this possible function of ORM1 in ameloblastomas, it is necessary to perform other experimental approaches and functional assays, focused on the elucidation of how these proteins may cooperate directly in the growth of this tumor.
In the present study, we report for the first time that Although we now have some information on the role of ORM1 in ameloblastoma and AC, many questions remain to be resolved. For example, although ORM1 expression was found in the analyzed samples, whether ORM1 expression was induced or merely increased in response to tumor growth or the onset of neoplastic cell production remains unknown. Additionally, the acidity and other properties of ORM1 may contribute to tissue degradation, thereby easing tumor growth.
Finally, although ORM1 is known to have many functions, immunological and functional experiments such as proliferation, migration and invasion assays or gene silencing and overexpression assays will be necessary to better elucidate the role of ORM1 in ameloblastomas.